This project seeks to discover small molecules active in inhibiting replication of Venezuelan Equine Encephalitis Viruses (VEEV), by utilizing a high throughput screening (HTS) campaign from the Molecular Library Program Center Network (MLPCN). VEEV, an encephalitic alphavirus, is listed as a select agent for its ability to cause severe disease during epidemics as well as its potential use a bioterror weapon. However, there is no FDA-approved treatment or prophylaxis of VEEV-related diseases at this time. Antiviral drug discovery for select agents has been impractical due to restrictions on handling the agents. Progress has been made toward the goal of efficacious treatment by developing a robust cytopathic effect (CPE)-based assay employing an attenuated VEEV strain, TC-83. The attenuated strain is not a select agent and can be handled in a BSL-2 lab. Because the attenuated strain has a significantly high homology between its genome sequence and the wild type VEEV, I hypothesize that the anti-viral compounds screened through this assay would be active for wild type VEEV as well. Thus the result of achieving the specific aims will be assays that can be used as a primary screen to discover active compounds from the Molecular Libraries Small Molecule Repository (MLSMR) library and the follow-up studies to verify activities for the attenuated and wild type virus strains. The first specific aim will be to screen the MLSMR library with a verified, cell-based assay measuring CPE caused by VEEV, strain TC-83 as a primary HTS. The second specific aim will be to verify the selected compounds from the primary screen in two ways 1) a dose-response cytotoxicity and efficacy assay and 2) re-screening with second attenuated strain, V3526 which has a higher similarity with wild types. Finally, a third battery of assays will be provided those are crucial for developing and characterizing chemical probes. The selected compounds will be subsequently verified using titer reduction assays with the wild-type strain, V3000. Last, for the compounds selected as probes, mode of action studies will be performed with reporter- based, molecular and biochemical assays. The screening pathway provided here may identify novel chemical scaffolds that can be pursued as therapeutics for encephalitic alphavirus infections including Eastern or Western equine encephalitis viruses. . Hence successful outcomes from this project will benefit the public and military, a goal that aligns with the MLPCN and the NIH. PUBLIC HEALTH RELEVANCE: Venezuelan Equine Encephalitis Virus (VEEV) is transmitted to humans and equine by mosquitoes and represents neuroinvasive diseasees. In general, disease is rare in US however epizootic strains of VEEV infected horses and approximately 70,000 ~ 100,000 people in South America in last epidemics in 1995 - 1996. There have also been reports of aerosol transmission of VEEV. VEEV has been weaponized and is a CDC/NIAID category B select agent for biodefense research. Despite of the importance in medical and bioterror needs, there are no treatments for VEEV infection. There is a need for new assays to identify compounds that inhibit VEEV replication. This work represents an unmet medical need for re-emerging viruses which has classified as a select agent. Of importance, Western, Eastern and Venezuelan equine encephalitis viruses are close in a phylogenetic analysis therefore successful outcomes from the proposed project will enlighten the way of discover potential therapeutics for important select agents which are closely related each other.